Mycobacterium tuberculosis (M.tb) infects one-third of the world's population and claims the lives of two to three million people each year. Its success is achieved by its ability to persist in the hostile intracellular environment of infected macrophages. After invasion, M.tb manipulates the phagocytic pathway of the host cell to inhibit the maturation of the phagosome into a phagolysosome. In addition, M.tb inhibits the apoptotic response of infected macrophages. These effects are likely to represent a highly evolved strategy that is used by M.tb to evade the host immune response. Although some cellular proteins have been characterized as targets of M.tb, it is currently unclear which bacterial proteins or lipids mediate the interactions. The proposed research project aims at filling this gap in our knowledge by focusing on the identification of proteins or lipids of M.tb that are implicated in either inhibition of maturation of the bacterial phagosome or inhibition of the apoptotic response of the host cell. The recent advances in the genetic manipulations of mycobacteria will be used to randomly mutagenize green fluorescence protein (GFP)-labeled M.tb and screen for mutants deficient in inhibiting phagosome maturation using a newly developed FACS-based, high- throughput assay. In a second approach, Mycobacterium smegmatis, a nonpathogenic mycobacterium deficient in inhibiting phagosome maturation and apoptosis of the host cell, will be complemented with M.tb genes and clones that have gained this capacity will be selected. Identification of M.tb genes essential for persistence of the pathogen will provide important targets for the development of new drugs for the treatment of tuberculosis and the development of new attenuated strains of M.tb that may be used as vaccines.